Nucleic acids encoding NGF variants

ABSTRACT

NGF variants which have trkC-binding activity and trkC-signal inducing activity are provided. The variants optionally have trkA or trkB binding and signal induction activity. The NGF variants of the present invention are useful in the treatment of neuronal disorders. Nucleic acids and expression vectors encoding the NGF variant neurotrophins are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/044,918, filed Apr. 25, 1997, the contents of whichare incorporated herein by reference.

BACKGROUND

1. Technical Field

This application relates to proteins which are involved in the growth,regulation or maintenance of nervous tissue, particularly neurons. Inparticular, it relates to NGF variants that have activities of otherneurotrophic factor NT-3. NGF variants which have trkC-binding activityand trkC-signal inducing activity are provided. The variants optionallyhave trkA or trkB binding and signal induction activity. The NGFvariants of the present invention are useful in the treatment ofneuronal disorders. Nucleic acids and expression vectors encoding theNGF variant neurotrophins are also provided.

2. Introduction

The survival and maintenance of differentiated function of vertebrateneurons is influenced by the availability of specific proteins referredto as neurotrophins. The neurotrophins form a highly homologous familyof growth factors that are important for survival and maintenance ofneurons during developmental and adult stages of the vertebrate nervoussystem (for review see Snider, 1994). Limited production ofneurotrophins results in death of superfluous neurons (for reviews, see(1); (2)). The various neurotrophins differ functionally in theirability to support survival of distinct neuronal populations in thecentral and the peripheral nerve system (3), (4); (5), (80).

The neurotrophin family is a highly homologous family which includesNT-3 (6), (7); (5); (8); (9); (10), nerve growth factor (NGF) (11);(12), brain-derived neurotrophic factor (BDNF) (13); (14)) andneurotrophin 4/5 (NT-4/5) ((15), (16), (17) and neurotrophin-6 (NT-6)(Barde, 1991; Götz et al., 1994).

Studies suggest that neurotrophins transduce intracellular signaling atleast in part through the ligand-dependent activation of a class oftyrosine kinase-containing receptors of M_(r)=140-145,000 known as thetrks (18); (19) (21); (20) (22); (23); (24); (25); (26). Binding of theneurotrophins induces autophosphorylation of the trk receptors whichtriggers the subsequent steps in the signal transduction cascade (Kaplan& Stephens, 1994). Thus, the signal transduction pathway ofneurotrophins is initiated by this high-affinity binding to andactivation of specific tyrosine kinase receptors and subsequent receptorautophosphorylation (19); (27). Although there is some degree ofcross-receptor interaction between the neurotrophins and the differenttrks, the predominant specificity appears to be NGF/trkA, BDNF/trkB, andNT-3/trkC while NT-4/5 appears to interact primarily with trkB asefficiently as BDNF (27); (19) (21); (25); (22); (28); (18); (28a). NGFinteracts exclusively with trkA (Kaplan et al., 1991) while BDNF andNT-4/5 bind to trkB (Ip et al., 1993). TrkA and trkB can respond invitro under certain circumstances to multiple neurotrophins (6); (23).TrkC responds exclusively to NT-3 (25); (26). NT-3 signals preferablythrough trkC but can also bind to trkA and trkB with lower affinity(Lamballe et al., 1991; Urfer et al., 1994) (FIG. 1). Thus, the moststringent member of the trk receptors in terms of specificity (trkC)interacts exclusively with the most promiscuous ligand (NT-3) of theneurotrophin family.

However, the neuronal environment does restrict trkA and trkB in theirability to respond to non-preferred neurotrophic ligands (29). Inaddition to the trk family of receptors, the neurotrophins can also bindto a different class of receptor termed the p75 low affinity NGFreceptor (p75; (30); (31)) which has an unknown mechanism oftransmembrane signaling but is structurally related to a receptor genefamily which includes the tumor necrosis factor receptor (TNFR), CD40,0X40, and CD27 (32); (33); (34), (35); (36); (37)). The role of the gp75in the formation of high-affinity binding sites and in the signaltransduction pathway of neurotrophins is as yet unclear (for reviews see(38); (39)).

An examination of the primary amino acid sequence of the neurotrophinsreveals seven regions of 7-10 residues each which account for 85% of thesequence divergence among the family members.

Nerve growth factor (NGF) is a 120 amino acid polypeptide homodimericprotein that has prominent effects on developing sensory and sympatheticneurons of the peripheral nervous system. NGF acts via specific cellsurface receptors on responsive neurons to support neuronal survival,promote neurite outgrowth, and enhance neurochemical differentiation.NGF actions are accompanied by alterations in neuronal membranes (40),(41), in the state of phosphorylation of neuronal proteins (42), (43),and in the abundance of certain mRNAs and proteins likely to play a rolein neuronal differentiation and function (see, for example (44)).

Forebrain cholinergic neurons also respond to NGF and may require NGFfor trophic support. (45). Indeed, the distribution and ontogenesis ofNGF and its receptor in the central nervous system (CNS) suggest thatNGF acts as target-derived neurotrophic factor for basal forebraincholinergic neurons (46), (81).

NT-3 transcription has been detected in a wide array of peripheraltissues (e. g. kidney, liver, skin) as well as in the central nervesystem (e. g. cerebellum, hippocampus) (5); (7), (82). Duringdevelopment, NT-3 mRNA transcription is most prominent in regions of thecentral nervous system in which proliferation, migration anddifferentiation of neurons are ongoing (50). Supporting evidence for arole in neuronal development includes the promoting effect of NT-3 onneural crest cells (51) and the stimulation of the proliferation ofoligodendrocyte precursor cells in vivo (79). NT-3 also supports invitro the survival of sensory neurons from the nodose ganglion (NG) (7);(5), (83) and a population of muscle sensory neurons from dorsal rootganglion (DRG) (52). In addition to these in vitro studies, a recentreport showed that NT-3 prevents in vivo the degeneration of adultcentral noradrenergic neurons of the locus coerulus in a model thatresembles the pattern of cell loss found in Alzheimer's disease.

Extensive mutational analyses of human NT-3 (Urfer et al., 1994) andmouse and human NGF (Ibáñez et al., 1993; Shih et al., 1994) suggestedthe binding sites for trkC and trkA, respectively. The three-dimensionalstructures of several neurotrophins have been resolved by X-raycrystallography (McDonald et al., 1991; Holland et al., 1994; Robinsonet al., 1995). In NGF the N-terminal residues contribute significantlyto affinity for trkA (Shih et al., 1994) and provide the most importantdeterminants for specificity (Ibáñez et al., 1993; Urfer et al., 1994).Significant losses of biological activity and receptor binding wereobserved with purified homodimers of human and mouse NGF, representinghomogenous truncated forms modified at the amino and carboxy termini(47); (48); (49). The 109 amino acid species (10-118)hNGF, resultingfrom the loss of the first 9 residues of the N-terminus and the last tworesidues from the C-terminus of purified recombinant human NGF, is300-fold less efficient in displacing mouse [¹²⁵I]NGF from the humantrkA receptor compared to (1-118)hNGF (49). It is 50- to 100-fold lessactive in dorsal root ganglion and sympathetic ganglion survivalcompared to (1-118)hNGF (48). The (1-118)hNGF has been reported to haveconsiderably lower trkA tyrosine kinase autophosphorylation activity(49).

For NT-3 it has been demonstrated that the epitope for trkC is formed byresidues in the central β-strand bundle region but does not includeresidues from non-conserved loops or the first six residues of theN-terminus (Urfer et al., 1994). However, a non-conserved β-hairpin loopencompassing residues 40-49 (NGF residue numbers will be used throughoutthe text) has been proposed to mediate trkA/trkC specificity (Ilag etal., 1994), though this loop does not contribute to NT-3 binding to trkC(Urfer et al., 1994). The mechanism of trkC discrimination, however, isunclear, especially since the most important residue in NT-3 involved inbinding to trkC, R103, is conserved in all neurotrophins.

The elucidation of the structural determinants for neurotrophinspecificity is important for understanding the function and evolution ofthis family of growth factors. Furthermore, administration ofneurotrophins in models of nerve lesions have been shown to bebeneficial for regeneration and survival of neurons (Sendtner et al.,1992; Yan et al., 1992). Since the neurotrophins have become candidatesfor therapeutics for a variety of neurodegenerative diseases, knowledgeof the structural mechanism of neurotrophic specificity and functionwill help develop novel neurotrophin-based therapeutics.

There has been some limited attempts to create chimeric orpan-neurotrophic factors. (See (53); (56); (54), (55)). Neuronalpopulations involved in neurodegenerative disorders may express morethan one trk receptor and therefore administration of molecules withmultiple specificities, such as MNTS-1 (Urfer et al., 1994) or PNT-1(Ibáñez et al., 1993) could be advantageous compared to administrationof a single monospecific neurotrophin or a cocktail of monospecificneurotrophins. For example, the various members of the neurotrophinfamily may have different pharmacokinetics and therefore the behavior ofneurotrophin cocktails could be difficult to predict or control.

There is a need for neurotrophic molecules that have more than oneneurotrophin activity and/or have improved pharmacokinetic propertiesand that are readily administered and retain effectiveness. These andother advantages are provide by the molecules and methods presentedherein.

SUMMARY

The present invention is based in part on the discovery that certainresidues that are part of the central β-strand bundle of NT-3 and arenot conserved among the neurotrophins can impart NT-3 trkC-binding andtrkC-signal inducing activity when grafted onto NGF. Exchange of NGFresidues at positions 18, 20, 23, 29, 84 and 86 by their NT-3counterparts resulted in NGF variants that bound to trkC, whilemaintaining their affinity to trkA, and were able to induceautophosphorylation and differentiation of PC12 cells expressing trkC.These NGF variants show that the amino acid at position 23 (Glycine inNGF/Threonine in NT-3) is critical for trkC recognition while otherresidues fine tune the specificity of NT-3 for trkC. The resultsdemonstrate the importance of non-conserved residues of the centralβ-strand bundle region for the interaction of NT-3 with trkC andemphasize the different mechanism of specificity determination that isemployed in the NT-3/trkC and NGF/trkA ligand/receptor pairs.

Accordingly, NGF variants are provided that have trkC-binding and signalinducing activity. The NGF variants optionally have trkA-binding andsignal induction activity and optionally have trkB-binding and signalinducing activity. In one embodiment the variant has both trkA and trkCactivity. In another embodiment, the variant has trkC activity but lackstrkA activity. The amino acid sequence of the NGF variants are derivedby the substitution, insertion or deletion of one or more amino acids ofan NGF amino acid sequence. Preferably, the NGF is a naturally-occurringmammalian NGF. Most preferably it is a human NGF. An NGF variant willtypically retain at least 75% amino acid sequence identity with the NGFparent molecule from which it is derived. Useful quantities of these NGFvariants are provided using recombinant DNA techniques.

It is a further aspect of the invention to provide recombinant nucleicacids encoding the NGF variants, and expression vectors and host cellscontaining these nucleic acids.

An additional aspect of the present invention provides methods forproducing the NGF variants, including methods using nucleic acid,vectors and host cells of the invention. In one embodiment a host celltransformed with an expression vector containing a nucleic acid encodingan NGF variant is cultured to allow expression of the nucleic acid toproduce a recombinant NGF variant.

Furthermore, methods and compositions for treating neuronal disorders ofa mammal are provided, which use the NGF variants of the invention.

Other aspects of the invention will become apparent from the followingdetailed description, the figures, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing specificities of neurotrophin/trk receptorinteractions.

FIG. 2 depicts a sequence alignment of human NGF (SEQ ID NO:1) and humanNT-3 (SEQ ID NO:2). Residue numbers that refer to the NGF sequence areused throughout the paper. Asterisks highlight NGF residues which weremutated in the variants analyzed in this study. Bars indicate locationsof β-strands in the X-ray structure of murine NGF (McDonald et al.,1991).

FIG. 3A depicts a model of human NT-3 and FIG. 3B shows the crystalstructure of murine NGF. The two monomers of each neurotrophin are shownin tan and gray; residue numbers in NGF gray monomer are denoted by a *.For highlighted residues, sidechain oxygen atoms are red and sidechainnitrogen atoms are blue. Residue 103 (Arg in both NGF and NT-3) ispurple. NGF residues which were replaced with their NT-3 counterpartsand affected binding and specificity are yellow; residues which did notaffect binding and specificity are green. The first residue seen in theNGF crystal structure (residue 10) is brown. The variable β-hairpin loop(residues 40-49) previously proposed to affect specificity (Ilag et al.,1994) is shown in cyan.

FIGS. 4A and 4B depict tyrosine phosphorylation of trkA in PC12 cellsexpressing rat trkC. Cells were treated with 100 ng/ml of respectiveneurotrophin for 5 min. Lysates were equalized for cell protein,immunoprecipitated with an anti-trkA specific polyclonal antiserum andelectrophoresed on 7.5% SDS-polyacryamide gels. Tyrosine phosphorylationwas detected using an anti-phosphotyrosine mAb 4G10. NGF/P, purifiedNGF; NGF/U, concentrated supernatant of NGF-expressing 293 cells;NT-3/P, purified NT-3; NT-3/U, concentrated supernatant of NT-3expressing 293 cells; 0, mock-treated 293 cells. FIGS. 4A and 4B showresults from two separate experiments using the neurotrophins listedabove each lane.

FIGS. 5A, 5B and 5C depict tyrosine phosphorylation of trkC in PC12cells expressing rat trkC. Cells were treated with 100 ng/ml ofrespective neurotrophin for 5 min. Lysates were equalized for cellprotein, immunoprecipitated with an anti-trkC specific antiserum 656 andelectrophoresed on 7.5% SDS-polyacryamide gels. Tyrosine phosphorylationwas detected using an anti-phosphotyrosine mAb 4G10. NGF/P, purifiedNGF; NGF/U, concentrated supernatant of NGF-expressing 293 cells;NT-3/P, purified NT-3; NT-3/U, concentrated supernatant of NT-3expressing 293 cells; 0, mock-treated 293 cells. FIGS. 5A, 5B and 5Cshow results from three separate experiments using the neurotrophinslisted above each lane.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

Single letter codes for the amino acids are used herein, as is known inthe art, according to the following Table 1:

TABLE 1 three letter single letter Amino acid abbreviation abbreviationAlanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp DCysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Serine Ser S Threonine Thr T Tryptophan Trp WTyrosine Tyr Y Valine Val V Proline Pro P

Thus, the identification of an amino acid residue is the single letteramino acid code followed by the position number of the residue. It is tobe understood that the position number corresponds to the particularneurotrophin backbone; thus, D15A NT3 means that the aspartic acid atposition 15 of NT3 is changed to an alanine. This aspartic acid, foundwithin a “constant region” as defined below, corresponds to position D16of NGF, since NGF has an additional amino acid at its N-terminus.

The present invention provides neurotrophic NGF variants havingtrkC-binding and signal inducing activity. Generally, a neurotrophin isa protein involved in the development, regulation and maintenance of thenervous system, and in particular of neurons. Currently, there areseveral known important neurotrophic factors: nerve growth factor (NGF),neurotrophin-3 (NT3), neurotrophin-4 (NT4, also sometimes calledneurotrophin-5 (NT5) or NT4/5), brain-derived neurotrophic factor(BDNF), ciliary neurotrophic factor (CNTF), and NT-6.

By the term “NGF variant” herein is meant a neurotrophin which, unlikenaturally occurring NGF, has neurotrophin specificity of NT-3 for trkCbinding and trkC signal induction. That is, the variant contains changesthat confer or impart these NT-3 activities. In one embodiment, thismeans that the NGF variant of the present invention will bind to atleast trkC, and optionally to variety of neurotrophic receptors,preferably trkA and/or trkB. Thus, for example, naturally occurring NGF,which is the natural or native ligand for the trkA receptor, does notbind appreciably to either the trkB or trkC receptor with high affinity;for example, NGF binds to these receptors with a 500-1000 fold lowerK_(D) than BDNF or NT-3, respectively. However, an NGF variant, i.e. aneurotrophin whose amino acid backbone is based on NGF, can bind to atleast the trkC. In one embodiment the NGF variant binds to trkC andtrkA, but not trkB. A preferred embodiment binds trkC but not trkA ortrkB. Alternatively, in one embodiment, the variant binds trkA, trkB,and trkC, as well as the p75 receptor. In another embodiment, thevariant binds trkC and trkB, but not trkA.

In one embodiment, the NGF variant binds to these receptors withaffinities higher than normally found, with affinities seen for thenatural ligand of that receptor. For example, NGF binds strongly totrkC, and very weakly or not at all to trkA and trkB. Thus, one NGFvariant embodiment binds to trkA with its normal or substantially normalbinding affinity, and will bind to either trkC with an affinitysubstantially similar to the trkC natural ligand, NT-3, or to trkB withan affinity similar to the trkB natural ligands BDNF or NT4/5, or both.

In the preferred embodiment, the binding affinity of the NGF variant forneurotrophin receptor trkC is less than about 50-fold lesser affinitythan NT-3 for trkC, preferably less than about 20-fold less, morepreferably less than about 15-fold less, even more preferably less thanabout 10-fold less, and even more preferred less than about 5 fold lessaffinity than NT-3 for trkC. In one embodiment, the NGF variant willhave about 3 fold less affinity for trkC than does NT-3. Variants canhave about the same or substantially the same affinity as NT-3, or evenhave less than or equal to about two fold higher activity than NT-3. Theabove affinity comparisons are also applied to NGF variant binding totrkB in comparison to BDNF.

This affinity is measured by a variety of ways, as will appreciated bythose skilled in the art. The preferred method is the use of competitionassays, as shown in (84) and in the Examples. Generally, bindingaffinities are reported as IC₅₀, that is, the concentration of unlabeledcompetitor which inhibits 50% of the binding of labeled ligand to thereceptor.

In alternative embodiments, the neurotrophin activity is measured not bybinding affinity to neurotrophin receptors, but rather by the neuronalsurvival or neurite outgrowth assays. This indicates the ability of avariant to induce trkC cellular signaling. Thus, all neurotrophinssupport the survival of embryonic neural crest-derived sensory neurons(77), (78), (7), (17). Survival of embryonic sympathetic neurons is onlysupported by NGF, while survival of placode-derived sensory neurons issupported by NT-3 and BDNF (85). Survival of sensory neurons of thedorsal root ganglion is supported by both NGF and BDNF (13). NT-3elicits neurite outgrowth of sensory neurons from dorsal root ganglion,sympathetic chain ganglia, and nodose ganglion, as well as supportssurvival of nodose ganglia neurons and dorsal root ganglion neurons.Thus, neuronal survival assays or neurite outgrowth assays can be run todetermine the activity of the NGF variant neurotrophins. Survival ofmotoneurons is another preferred NGF variant activity.

In one preferred embodiment, the activity of an NGF variant isdetermined by its ability to stimulate differentiation of PC12 cellsexpressing trkC. PC12 cells expressing trkC (Tsoulfas et al., 1993;Tsoulfas et al., 1996) can be plated onto collagen-coated tissue culturedishes, and assayed for the proportion of neurite-bearing cells. Aneurite bearing cell is defined as one containing processes at leasttwice the length of the cell body after 3 days. Alternatively, PC12cells expressing trkB can be used to test for trkB signal inducingactivity. A preferred NGF variant will achieve a neurite outgrowth atleast about 30% the maximal response of NT-3, more preferably about 50%,even more preferably about 75%, yet more preferably about 90%, and mostpreferably about 100% maximal NT-3 response, when compared at the sameconcentration in the medium, and most preferably when compared at theconcentration of NT-3 needed for maximal response. For example, in Table4, preferred comparisons are made at 1 nanogram of factor per ml ofmedium.

Thus, neurotrophin specificity is determined by the neurotrophinreceptor binding, and the neuronal survival assays and/or neuriteoutgrowth assays. Thus, an NGF variant as defined herein is aneurotrophin NGF which exhibits at least the binding characteristics,neuronal survival assay specificity, or the neurite outgrowth assayspecificity of NT-3. Optionally, a NGF variant with BDNF or NT4/5specificity exhibits at least the binding characteristics, neuronsurvival assay specificity, or neurite outgrowth assay specificity ofBDNF or NT4/5, respectively, with the preferences as discussed for NT-3activity, e.g., neurite outgrowth at least about 30% the maximalresponse of BDNF, more preferably about 50%, etc.

NGF variants are that have trkC-binding and signal inducing activitywill typically retain at least 75% amino acid sequence identity with theNGF parent molecule from which it is derived. Preferably at least 80%identity, more preferably at least 90% identity, and most preferably atleast 95% identity with the the NGF parent molecule from which it isderived.

Since the trkC binding site on NT-3 is dominated by residues in thecentral β-strand bundle region, it was hypothesized that trkC recognizesthis set of residues to distinguish NT-3 from the other neurotrophins(Urfer et al., 1994). In the present work, a set of five residueslocated in this structural region was transferred from human NT-3 tohuman NGF (FIG. 2). The resulting NGF variant G23T/V18E/V20L/T81K/H84Q(NGF12) bound to trkC, maintained its affinity to trkA, and stimulatedautophosphorylation and differentiation of PC12 cells expressing trkC.Further mutagenesis revealed that the most important determinants forspecific trkC binding are located at positions 23 and 84 and thatresidues at positions 18, 20, 29 and 86 fine tune specificity for trkC.These results demonstrate the importance of non-conserved residues ofthe central β-strand bundle region for the interaction of NT-3 withtrkC, emphasize the different mechanism of specificity determinationthat is employed in the NT-3/trkC and NGF/trkA ligand/receptor pairs,and support the proposal that the overall structure of neurotrophins, incontrast to short amino acid “active-site” segments, may determineneurotrophin specificity (Suter et al., 1992).

An NGF variant of the invention can be derived from NGF by substitutingat amino acid positions G23, H84, and V18 or V20 to impart trkC bindingin order to obtain an NGF variant that binds trkC. In a preferredembodiment the V20 is substituted. Optionally, the NGF variant canfurther contain a substitution of F86, T81, or T29, which can furtherenhance or fine tune NT-3 specificity. The trkC-binding or signalinducing activity is recruited or imparted by changes in NGF thatconsist essentially of the changes discussed herein. Preferred NGFvariants include NGF130, NGF131, NGFR2, and NGFR3, as shown in theExamples. Most preferred substitutions at these positions are G23T,H84Q, V18E, V20L, F86Y, T81K, and T29I. However, other substitutions areappropriate, preferably conservative or homolog series substitutions.For example, V18 can be conservatively substituted with leucine orthreonine to minimally effect trkC binding or a glutamic acidconservative amino acid or homolog, such as with aspartic acid orglutamine, to enhance trkC binding. V20 can be substituted with largerhydrophobic amion acids isoleucine, methionine, or threonine to enhancetrkC binding, and perhaps alanine to have a minimal trkC binding effect.G23 can be substituted with serine. T29 can be substituted withisoleucine, valine, leucine or serine. Preferably, T29I is present whenF86Y is present. Y79 can be substituted with glutamine, phenylalanine,or asparagine, but preferably remains Y79. T81 can be substituted witharginine to effect trkC binding or with isoleucine, valine, leucine orserine with minimal trkC binding effect. H84 can be substituted withasparagine. F86 can be substituted with methionine, tryptophan,threonine or serine to effect trkC binding.

The NGF variant can further contain a modification of the NGF sequencethat imparts trkB binding to yield an NGF variant that also binds trkBin addition to trkC. A preferred modification is the amino acidsubstitution at D16. A preferred variant has D16A substitution.WO/95/33829, published Dec. 14, 1995, which discloses D16A NGF, isincorporated herein by reference. Other alanine conservativesubstitutions at D16 can be made to obtain trkB binding. In someembodiments, there is more than one domain within a neurotrophin whichcan confer neurotrophic specificity, which will depend on the particularneurotrophin. BDNF, for example, has a number of domains which appear toconfer BDNF specificity. For example, the substitution of the BDNFsequence from positions 93-99 (SKKRIG) may confer BDNF specificity (55)in NGF. will depend on the particular neurotrophin. BDNF, for example,has a number of domains which appear to confer BDNF specificity. Forexample, the substitution of the BDNF sequence from positions 93-99(SKKRIG) (SEQ ID NO:14) may confer BDNF specificity (55) in NGF.

NGF has a number of domains which can affect NGF specificity whenmodified. The N-terminal amino acids of NGF are the main region in NGFresponsible for trkA binding. Significant losses of biological activityand receptor binding were observed with purified homodimers of human andmouse NGF, representing homogenous truncated forms modified at the aminoand carboxy termini. The 109 amino acid species (10-118)hNGF, resultingfrom the loss of the first 9 residues of the N-terminus and the last tworesidues from the C-terminus of purified recombinant human NGF, is300-fold less efficient in displacing mouse [¹²⁵I]NGF from the humantrkA receptor compared to (1-118)hNGF. It is 50- to 100-fold less activein dorsal root ganglion and sympathetic ganglion survival compared to(1-118)hNGF. The NGF variant can contain a modification of the10-amino-acid-N-terminal region to reduce or eliminate trkA binding. Inone embodiment, the 7 N-terminal amino acids (SSSHPIF) (SEQ ID NO:15) ofNGF can be deleted or substituted, for example, with the N-terminalamino acids of NT-3 (YAEHKS) (SEQ ID NO:16), to obtain an NGF variantwith reduced or absent trkA-binding activity. The exact number of NGFN-terminal residues modified is not crucial, with from about 4 to about10 N-terminal residues may be exchanged, although in some embodiments, asingle amino acid change will be sufficient. In one embodiment at leastone of the 10 N-terminal amino acids are deleted or substituted toreduce eliminate trkA binding. A particularly preferred position formodification is the histidine at amino acid position 4, which appears tobe quite important for NGF specificity, as well as the proline atposition 5. Similarly, a number of other residues of NGF have been shownto be important in NGF trkA receptor binding as well as bioactivityassays. For example, there are a number of residues which, when mutated,lose NGF activity. These residues include, but are not limited to, D30,Y52, R59, R69, and H75. While alanine can be substituted at thesepositions to disrupt trkA binding, other amino acids that arenon-conservative to the amion acid at that position can also be used. WO95/33829, published Dec. 14, 1995, which discloses NGF variants thatlack NGF activity, is incorporated herein by reference. ThetrkB-recruiting modification can be combined with the trkA-reducingmodification to yield a variant that binds both trkC and trkB, but nottrkA.

Also provided are NGF variants containing an NGF having substitutions atamino acid positions V18, V20, G23, H84 and either F86 or T81 or boththat impart trkC binding, wherein the variant binds trkC. In a preferredembodiment, these NGF variants are substituted at both T81 and F86 toenhance or fine tune the NT-3 activity. These NGF variants can furthercontain a substitution of T29. Most preferred substitutions at thesepositions are G23T, H84Q, V18E, V20L, F86Y, T81K, and T29I. PreferredNGF variants of this type include NGF126, NGF1234, NGF124, NGF125,NGF12, and NGF123, and NGF127, as shown in the Examples. Othersubstitutions at these positions as discussed herein are also suitable.

NGF is a 120 amino acid polypeptide homodimeric protein. While NGF canbe produced in its 120 form, a more preferred parent or backbone formfor NGF variants is the 118 amino acid form, preferably in dimer form.In the 118 form R119 and A120 are absent. This form can be obtained byexpression using a 118-NGF-encoding nucleic acid or by selectivepost-translational proteolysis of the 120 form, e.g., with trypsin. Inanother embodiment the 117 NGF form serves as the parent or backboneNGF. A composition containing an NGF variant or variants and aphysiologically acceptable carrier are also provided.

In addition, residues in the vicinity of the residues discussed abovecan also enhance or fine tune NT-3 specificity. In some embodiments,changes in the constant regions may also give NT3 specificity.Alternatively, mutations at positions R31 and E92 in NT-3, which causeincreases in NT-3 binding to trkC, specifically, R31A and E92A NT3 canbe incorporated into the corresponding positions in NGF, using theprocedures described below.

Furthermore, there are a number of amino acid substitutions in NGF whichincrease NGF binding and/or bioactivity. Accordingly, thesesubstitutions may be included in the NGF variant backbones to enhanceNGF specificity. These residues include, but are not limited to, E11,F12, D24, E41, N46, S47, K57, D72, N77, D105, and K115. While alaninecan be substituted at these position to maintain or enhance trkAbinding, other amino acids that are conservative to alanine or to theamino acid at that position can also be used. The following provides aguideline.

The residues important in neurotrophin specificity can be replaced byany of the other amino acid residues using techniques described in theexamples and well-known techniques for site-directed mutagenesis.Generally, the amino acids to be substituted are chosen on the basis ofcharacteristics understood by those skilled in the art. For example,when small alterations in the characteristics are desired, substitutionsare generally made in accordance with the following table:

TABLE 2 Exemplary Original Residue Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr SerTrp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTable 1. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. In a preferredembodiment, the residues are changed to alanine residues.

In addition, homologue-scanning mutagenesis, random mutagenesis,cassette mutagenesis, can all be used to generate putative NGF variantswhich may then be screened for receptor binding using the techniquesdescribed in the Examples and well-known in the art.

By the term “neurotrophin receptor” or grammatical equivalents herein ismeant a receptor which binds a neurotrophin ligand. In some embodiments,the neurotrophin receptor is a member of the tyrosine kinase family ofreceptors, generally referred to as the “trk” receptors, which areexpressed on the surface of distinct neuronal populations. The trkfamily includes, but is not limited to, trkA (also known as p140^(trk));trkB (also known as p145^(trkB)); and trkC (also known as p145^(trkC)).In other embodiments, the neurotrophin receptor is p75^(NGFR), alsocalled p75 or low-affinity nerve growth factor receptor (LNGFR). It isto be understood that other as yet undiscovered neurotrophin receptorsmay also bind the NGF variant neurotrophins of the present invention, aswill be easily ascertainable by those skilled in the art.

In one embodiment, binding to the p75 receptor by the NGF variant hasbeen substantially diminished or eliminated. For example, there are avariety of amino acid residues which contribute to p75 binding, in whichmutations result in diminished p75 binding. In NGF, mutations atpositions F12, I31, K32, K34, K50, Y52, R69, K74, K88, L112, S113, R114,and K115 all result in diminished p75 binding. F12, I31, K50, Y52, R69,and K74 are all within constant regions.

In addition to the amino acid changes outlined above, those skilled inthe art understand that some variability of the amino acid sequence istolerated without altering the specificity and characteristics of theneurotrophin. Thus, NGF variants can have amino acid substitutions,insertions or deletions compared to the wild-type sequences which do notaffect trk binding but are merely variations of the sequence. In someembodiments, these mutations will be found within the same positionsidentified as important to specificity; i.e. in some cases, neutralmutations may be made without changing neurotrophin specificity.

The NGF variant neurotrophins of the present invention can be made in avariety of ways, using recombinant technology. By the term “recombinantnucleic acid” herein is meant nucleic acid in a form not normally foundin nature. That is, a recombinant nucleic acid is flanked by anucleotide sequence not naturally flanking the nucleic acid or has asequence not normally found in nature. Recombinant nucleic acids can beoriginally formed in vitro by the manipulation of nucleic acid byrestriction endonucleases, or alternatively using such techniques aspolymerase chain reaction. It is understood that once a recombinantnucleic acid is made and reintroduced into a host cell or organism, itwill replicate non-recombinantly, i.e., using the in vivo cellularmachinery of the host cell rather than in vitro manipulations; however,such nucleic acids, once produced recombinantly, although subsequentlyreplicated non-recombinantly, are still considered recombinant for thepurposes of the invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e., through the expression of a recombinant nucleic acidas depicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated away from some or all of the proteins andcompounds with which it is normally associated in its wild type host.The definition includes the production NGF variant neurotrophins fromone organism in the same or different organism or host cell. Forexample, the protein may be made in the same organism from which it isderived but at a significantly higher concentration than is normallyseen, e.g., through the use of a inducible or high expression promoter,such that increased levels of the protein is made. Alternatively, theprotein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions anddeletions.

Using the nucleic acids of the invention which encode NGF variantneurotrophins, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the NGF variantneurotrophin. “Operably linked” in this context means that thetranscriptional and translational regulatory DNA is positioned relativeto the coding sequence of the NGF variant neurotrophin in such a mannerthat transcription is initiated. Generally, this will mean that thepromoter and transcriptional initiation or start sequences arepositioned 5′ to the NGF variant neurotrophin coding region. Thetranscriptional and translational regulatory nucleic acid will generallybe appropriate to the host cell used to express the NGF variantneurotrophin; for example, transcriptional and translational regulatorynucleic acid sequences from mammalian cells will be used to express theNGF variant neurotrophin in mammalian cells. Numerous types ofappropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, signalsequences, ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, termination and polyA signal sequences, and enhancer or activator sequences. In a preferredembodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences. Methods, vectors, and hostcells suitable for adaptation to the synthesis of NGF variants inrecombinant vertebrate cell culture are described in Gething et al.,Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP117,060; and EP 117,058. A particularly useful plasmid for mammaliancell culture expression of an NGF variant is pRK5 (EP 307,247), pRK7, orpSVI6B. WO 91/08291 published Jun. 13, 1991, is incorporated herein byreference.

Promoter sequences encode either constitutive or inducible promoters.Hybrid promoters, which combine elements of more than one promoter, arealso known in the art, and are useful in the invention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammaliancells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

The NGF variant neurotrophins of the invention are produced by culturinga host cell transformed with an expression vector containing nucleicacid encoding a NGF variant neurotrophin, under the appropriateconditions to induce or cause expression of the NGF variantneurotrophin. The conditions appropriate for NGF variant neurotrophinexpression will vary with the choice of the expression vector and thehost cell, and will be easily ascertained by one skilled in the art. Forexample, the use of constitutive promoters in the expression vector willrequire optimizing the growth and proliferation of the host cell, whilethe use of an inducible or repressible promoter requires the appropriategrowth conditions for induction or derepression.

In a preferred embodiment, the NGF variant neurotrophin is purified orisolated after expression. The NGF variant neurotrophins may be isolatedor purified in a variety of ways known to those skilled in the artdepending on what other components are in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. For general guidance in suitablepurification techniques, see (57). The degree of purification necessarywill vary depending on the use of the NGF variant neurotrophin. In someinstances no purification will be necessary.

Appropriate host cells include yeast, bacteria, archebacteria, fungisuch as filamentous fungi, and plant and animal cells, includingmammalian cells. Of particular interest are Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, Pichia pastoris, SF9 cells,C129 cells, 293 cells, Neurospora, and CHO, COS, HeLa cells,immortalized mammalian myeloid and lymphoid cell lines. A preferred hostcell is a mammalian cell, and the most preferred host cells include CHOcells, COS-7 cells, and human fetal kidney cell line 293.

In a preferred embodiment, the NGF variant neurotrophins of theinvention are expressed in mammalian cells. Mammalian expression systemsare also known in the art.

Some genes may be expressed more efficiently when introns are present.Several cDNAs, however, have been efficiently expressed from vectorsthat lack splicing signals. Thus, in some embodiments, the nucleic acidencoding the NGF variant neurotrophin includes introns.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used, and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

In one embodiment, NGF variant neurotrophins are produced in yeastcells. Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. The methods of introducing exogenous nucleic acidinto yeast hosts, as well as other hosts, is well known in the art, andwill vary with the host cell used.

In a preferred embodiment, NGF variant neurotrophins are expressed inbacterial systems. Expression vectors for bacteria are well known in theart, and include vectors for Bacillus subtilis, E. coli, Streptococcuscremoris, and Streptococcus lividans, among others. The bacterialexpression vectors are transformed into bacterial host cells usingtechniques well known in the art, such as calcium chloride treatment,electroporation, and others.

In one embodiment, NGF variant neurotrophins are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form; for example the “MaxBac”kit from Invitrogen in San Diego.

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for Aedes aegypti, Autographacalifornica, Bombyx mori, Drosophila melangaster, Spodoptera frugiperda,and Trichoplusia ni.

Once expressed, NGF variant neurotrophins are used as neurotrophicfactors. These NGF variant neurotrophins may be utilized in variousdiagnostic and therapeutic applications. The NGF variants can also beused as animal feed.

The NGF variant neurotrophins of the present invention are useful indiagnostic methods of detecting neurotrophin receptors. For example, theNGF variant neurotrophins of the present invention may be labelled. By a“labelled NGF variant neurotrophin” herein is meant a NGF variantneurotrophin that has at least one element, isotope or chemical compoundattached to enable the detection of the NGF variant neurotrophin or theNGF variant neurotrophin bound to a neurotrophin receptor. In general,labels fall into three classes: a) isotopic labels, which may beradioactive or heavy isotopes; b) immune labels, which may be antibodiesor antigens; and c) colored or fluorescent dyes. The labels may beincorporated into the NGF variant neurotrophin at any position. Oncelabelled, the NGF variant neurotrophins are used to detect neurotrophinreceptors, either in vitro or in vivo. For example, the presence ofneurotrophin receptors can be an indication of the presence of certaincell types, useful in diagnosis. That is, a subpopulation of certaincell types may be shown by the binding of the labelled NGF variantneurotrophin to the cells via the receptors.

Additionally, the NGF variant neurotrophins of the present invention areuseful as standards in neurotrophin assays. For example, the activity ofa NGF variant neurotrophin in any particular assay may be determinedusing known neurotrophin standards, and then the NGF variantneurotrophin may be used in the diagnosis and quantification ofneurotrophins.

Furthermore, the NGF variant neurotrophins of the present invention areuseful as components of culture media for use in culturing nerve cellsin vivo, since many nerve cell cultures require growth factors. As willbe understood by those skilled in the art, the NGF variant neurotrophinsof the present invention can replace other neurotrophic factors whichare frequently used as media components. The amount of the NGF variantneurotrophins to be added can be easily determined using standardassays.

The NGF variant neurotrophins of the present invention are also usefulto generate antibodies, which can be used in the diagnosis,identification, and localization of neurotrophins or neurotrophinantibodies within an organism or patient. For example, the NGF variantneurotrophins can be used to make polyclonal or monoclonal antibodies asis well known by those skilled in the art. The antibodies can then belabelled and used to detect the presence, or absence, of theneurotrophins. Thus, diagnosis of neural disorders associated withneurotrophins may be detected. Alternatively, the antibodies aredetected indirectly, by using a second antibody. For example, primaryantibodies may be made in mice or rabbits, and then labelled anti-mouseor anti-rabbit antibodies are used to detect the primary antibodies.Either of these methods, as well as similar methods well known in theart, allow the detection of neurotrophins in a variety of tissues.

In addition, the antibodies generated to the NGF variant neurotrophinsof the present invention are also useful for the purification ofneurotrophins and NGF variant neurotrophins. Since generally the aminoacid substitutions of the NGF variant neurotrophins are small, manyimmune epitopes are shared by the neurotrophins and NGF variantneurotrophins. Thus, antibodies generated to the NGF variantneurotrophins will bind naturally occurring neurotrophins, and thus areuseful in purification. For example, purification schemes based onaffinity chromatography techniques can be used, as are well known in theart.

NGF variant formulations of the invention are believed to be useful inpromoting the development, maintenance, or regeneration of neurons invitro and in vivo, including central (brain and spinal chord),peripheral (sympathetic, parasympathetic, sensory, and enteric neurons),and motor neurons. Accordingly, NGF variant formulations of theinvention are utilized in methods for the treatment of a variety ofneurologic diseases and disorders. In a preferred embodiment, theformulations of the present invention are administered to a patient totreat neural disorders. By “neural disorders” herein is meant disordersof the central and/or peripheral nervous system that are associated withneuron degeneration or damage. Specific examples of neural disordersinclude, but are not limited to, Alzheimer's disease, Parkinson'sdisease, Huntington's chorea, stroke, ALS, peripheral neuropathies, andother conditions characterized by necrosis or loss of neurons, whethercentral, peripheral, or motor neurons, in addition to treating damagednerves due to trauma, bums, kidney disfunction, injury, and the toxiceffects of chemotherapeutics used to treat cancer and AIDS. For example,peripheral neuropathies associated with certain conditions, such asneuropathies associated with diabetes, AIDS, or chemotherapy may betreated using the formulations of the present invention. It also isuseful as a component of culture media for use in culturing nerve cellsin vitro or ex vivo.

In various embodiments of the invention, NGF variant formulations areadministered to patients in whom the nervous system has been damaged bytrauma, surgery, stroke, ischemia, infection, metabolic disease,nutritional deficiency, malignancy, or toxic agents, to promote thesurvival or growth of neurons, or in whatever conditions are treatablewith NGF, NT-3, BDNF or NT4-5. As would be expected, the treatment oreffect is dependent on the particular trk-binding function or functionspresent in the NGF variant. For example, NGF variant formulation of theinvention can be used to promote the survival or growth of motor neuronsthat are damaged by trauma or surgery. Also, NGF variant formulations ofthe invention can be used to treat motoneuron disorders, such asamyotrophic lateral sclerosis (Lou Gehrig's disease), Bell's palsy, andvarious conditions involving spinal muscular atrophy, or paralysis. NGFvariant formulations of the invention can be used to treat humanneurodegenerative disorders, such as Alzheimer's disease, Parkinson'sdisease, epilepsy, multiple sclerosis, Huntington's chorea, Down'sSyndrome, nerve deafness, and Meniere's disease. NGF variantformulations of the invention can be used as cognitive enhancer, toenhance learning particularly in dementias or trauma. Alzheimer'sdisease, which has been identified by the National Institutes of Agingas accounting for more than 50% of dementia in the elderly, is also thefourth or fifth leading cause of death in Americans over 65 years ofage. Four million Americans, 40% of Americans over age 85 (the fastestgrowing segment of the U.S. population), have Alzheimer's disease.Twenty-five percent of all patients with Parkinson's disease also sufferfrom Alzheimer's disease-like dementia. And in about 15% of patientswith dementia, Alzheimer's disease and multi-infarct dementia coexist.The third most common cause of dementia, after Alzheimer's disease andvascular dementia, is cognitive impairment due to organic brain diseaserelated directly to alcoholism, which occurs in about 10% of alcoholics.However, the most consistent abnormality for Alzheimer's disease, aswell as for vascular dementia and cognitive impairment due to organicbrain disease related to alcoholism, is the degeneration of thecholinergic system arising from the basal forebrain (BF) to both thecodex and hippocampus (Bigl et al. in Brain Cholinergic Systems, M.Steriade and D. Biesold, eds., Oxford University Press, Oxford,pp.364-386 (1990)). And there are a number of other neurotransmittersystems affected by Alzheimer's disease (Davies Med. Res. Rev.3:221(1983)). However, cognitive impairment, related for example todegeneration of the cholinergic neurotransmitter system, is not limitedto individuals suffering from dementia. It has also been seen inotherwise healthy aged adults and rats. Studies that compare the degreeof learning impairment with the degree of reduced cortical cerebralblood flow in aged rats show a good correlation (Berman et al.Neurobiol. Aging 9:691 (1988)). In chronic alcoholism the resultantorganic brain disease, like Alzheimer's disease and normal aging, isalso characterized by diffuse reductions in cortical cerebral blood flowin those brain regions where cholinergic neurons arise (basal forebrain)and to which they project (cerebral cortex) (Lofti et al., Cerebrovasc.and Brain Metab. Rev 1:2 (1989)). Such dementias can be treated byadministration of NGF variant formulations of the invention.

Further, NGF variant formulations of the invention are preferably usedto treat neuropathy, and especially peripheral neuropathy. “Peripheralneuropathy” refers to a disorder affecting the peripheral nervoussystem, most often manifested as one or a combination of motor, sensory,sensorimotor, or autonomic neural dysfunction. The wide variety ofmorphologies exhibited by peripheral neuropathies can each be attributeduniquely to an equally wide number of causes. For example, peripheralneuropathies can be genetically acquired, can result from a systemicdisease, or can be induced by a toxic agent. Examples include but arenot limited to diabetic peripheral neuropathy, distal sensorimotorneuropathy, or autonomic neuropathies such as reduced motility of thegastrointestinal tract or atony of the urinary bladder. Examples ofneuropathies associated with systemic disease include post-poliosyndrome or AIDS-associated neuropathy; examples of hereditaryneuropathies include Charcot-Marie-Tooth disease, Refsum's disease,Abetalipoproteinemia, Tangier disease, Krabbe's disease, Metachromaticleukodystrophy, Fabry's disease, and Dejerine-Sottas syndrome; andexamples of neuropathies caused by a toxic agent include those caused bytreatment with a chemotherapeutic agent such as vincristine, cisplatin,methotrexate, or 3′-azido-3′-deoxythymidine. Accordingly, a method oftreating a neural disorder in a mammal comprising administering to themammal a therapeutically effective amount of an NGF variant is provided.Preferably, the neural disorder is a peripheral neuropathy, morepreferably diabetic peripheral neuropathy, chemotherapy-inducedperipheral neuropathy, or HIV-associated neuropathy. Preferably theperipheral neuropathy affects motor neurons.

Additionally, the administration of NT-3 prevents the in vivodegeneration of adult central noradrenergic neurons of the locuscoerulus in a model that resembles the pattern of cell loss found inAlzheimer's disease (86) In addition, the addition of NT3 has been shownto enhance sprouting of corticospinal tract during development, as wellas after adult spinal cord lesions (58). In fact, when NT3 wasadministered with antibodies which inhibit myelin-associated growthinhibitory proteins, long-distance regeneration was seen. Thus, the NGFvariant neurotrophins of the present invention can be used in place ofNT3 in this application.

In this embodiment, a therapeutically effective dose of a NGF variantneurotrophin is administered to a patient. By “therapeutically effectivedose” herein is meant a dose that produces the therapeutic effects forwhich it is administered. The exact dose will depend on the disorder tobe treated, and will be ascertainable by one skilled in the art usingknown techniques. In general, the NGF variant neurotrophins of thepresent invention are administered at about 1 μg/kg to about 100 mg/kgper day. In addition, as is known in the art, adjustments for age aswell as the body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

In some embodiments, the compositions are prepared containing amounts ofNGF variant from 0.07 to 20 mg/ml, preferably 0.08 to 15 mg/ml, morepreferably 0.09 to 10 mg/ml, and most preferably 0.1 to 2 mg/ml. For useof these compositions in administration to human patients suffering fromperipheral neuropathies, for example, these compositions may containfrom about 0.1 mg/ml to about 2 mg/ml NGF variant, corresponding to thecurrently contemplated NGF dosage rate for such treatment. NGF variantis expected to be well-tolerated, as is NGF, and higher doses can beadministered if necessary as determined by the physician. Since a singleIV or SC dose of 1 ug/kg NGF has been observed to cause injection sitehyperalgesia and total or partial body myalgias in human patients,preferred single dose IV or SC is preferably less than 1 ug/kg of NGF,although higher doses can be given when steps are taken to amelioratethe hyperalgesia and myalgias, as would be known in the art. A preferredregimen for peripheral neuropathy is 0.1 ug/kg, three times per week SCor 0.3 ug/kg, once weekly SC.

A “patient” for the purposes of the present invention includes bothhumans and other animals and organisms. Thus the methods are applicableto both human therapy and veterinary applications.

The administration of the NGF variant neurotrophins of the presentinvention can be done in a variety of ways, e.g., those routes known forspecific indications, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraarterially, intralesionally,intraventricularly in the brain, or intraocularly. The NGF variantneurotrophins may be administered continuously by infusion into thefluid reservoirs of the CNS, although bolus injection is acceptable,using techniques well known in the art, such as pumps or implantation.In some instances, for example, in the treatment of wounds, the NGFvariant neurotrophins may be directly applied as a solution or spray.Sustained release systems can be used. Generally, where the disorderpermits, one should formulate and dose the NGF variant for site-specificdelivery. Administration can be continuous or periodic. Administrationcan be accomplished by a constant- or programmable-flow implantable pumpor by periodic injections.

Semipermeable, implantable membrane devices are useful as means fordelivering drugs in certain circumstances. For example, cells thatsecrete soluble NGF variant can be encapsulated, and such devices can beimplanted into a patient, for example, into the brain or spinal chord(CSF) of a patient suffering from Parkinson's Disease. See, U.S. Pat.No. 4,892,538 of Aebischer et al.; U.S. Pat. No. 5,011,472 of Aebischeret al.; U.S. Pat. No. 5,106,627 of Aebischer et al.; PCT Application WO91/10425; PCT Application WO 91/10470; Winn et al., Exper. Neurology,113:322-329 (1991); Aebischer et al., Exper. Neurology, 111:269-275(1991); and Tresco et al., ASAIO, 38:17-23 (1992). Finally, the presentinvention includes an implantation device, for preventing or treatingnerve damage or damage to other cells as taught herein, containing asemipermeable membrane and a cell that secretes NGF variant encapsulatedwithin the membrane, the membrane being permeable to NGF variant, andimpermeable to factors from the patient detrimental to the cells. Thepatient's own cells, transformed to produce NGF variant ex vivo, couldbe implanted directly into the patient, optionally without suchencapsulation. The methodology for the membrane encapsulation of livingcells is familiar to those of ordinary skill in the art, and thepreparation of the encapsulated cells and their implantation in patientsmay be accomplished readily as is known in the art. Preferably, thesecreted NGF variant is a human mature NGF backbone variant when thepatient is human. The implants are preferably non-immunogenic and/orprevent immunogenic implanted cells from being recognized by the immunesystem. For CNS delivery, a preferred location for the implant is thecerebral spinal fluid of the spinal cord.

The pharmaceutical compositions of the present invention comprise a NGFvariant neurotrophin in a form suitable for administration to a patient.In the preferred embodiment, the pharmaceutical compositions are in awater soluble form, and may include such physiologically acceptablematerials as carriers, excipients, stabilizers, buffers, salts,antioxidants, hydrophilic polymers, amino acids, carbohydrates, ionic ornonionic surfactants, and polyethylene or propylene glycol. The NGFvariant neurotrophins may be in a time-release form for implantation, ormay be entrapped in microcapsules using techniques well known in theart.

NGF variant is preferably formulated in physiologically acceptableacetate buffer from pH 5 to pH 6, to markedly increase stability inthese compositions. Acetate concentrations can range from 0.1 to 200 mM,more preferably from 1 to 50 mM, and even more 5 to 30 mM, and mostpreferably from 10 to 20 mM. One preferred embodiment has 20 mM acetateand another has 10 mM acetate in the solution. A preferred acetate saltfor enhancing stability and buffering capacity is sodium acetate.However other physiologically acceptable acetate salts can be used, forexample potassium acetate. Suitable pH ranges for the preparation of thecompositions herein are from 5 to 6, preferably 5.4 to 5.9, morepreferably 5.5 to 5.8. A preferred pH is 5.5 which enhances stabilityand buffering capacity. Another preferred embodiment is pH 5.8.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Low concentrations are preferred, e.g.,less than about 0.3 M to about .05 M, preferably from 0.16 to 0.20 MNaCl, more preferably 0.13 to 0.15 M. In a preferred embodiment thesodium chloride concentration is 136 mM. In another preferred embodimentthe concentration is 142 mM.

Optionally, the formulations of the invention can contain apharmaceutically acceptable preservative. In some embodiments thepreservative concentration ranges from 0.1 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts.Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben arepreferred preservatives. Benzyl alcohol is a particularly preferredpreservative that results in enhanced NGF stability. A particularlypreferred benzyl alcohol concentration is 0.7 to 1.2%, more preferably0.8 to 1.0%, with a particularly preferred concentration of 0.9%.

Optionally, the formulations of the invention can include apharmaceutically acceptable surfactant. Preferred surfactants arenon-ionic detergents. Preferred surfactants include Tween 20 andpluronic acid (F68). F68 is particularly preferred for enhancing NGFvariant stability. Suitable surfactant concentrations are 0.005 to0.02%. A preferred concentration for surfactant is 0.01%. Surfactantsare used to minimize particulate formation.

In a particularly preferred embodiment the composition contains an NGFvariant concentration of 0.1 mg/ml, a sodium acetate concentration of 20mM, pH 5.5, a sodium chloride concentration of 136 mM, and benzylalcohol concentration at 0.9% (v/v). In another embodiment the NGFvariant concentration is 2.0 mg/ml, the sodium acetate concentration is10 mM, pH 5.5, and the sodium chloride concentration is 142 mM.

In another embodiment of the invention is provided a kit for NGF variantadministration, which includes a vial or receptacle containing apharmaceutical composition of the invention comprising apharmaceutically effective amount of NGF variant and a pharmaceuticallyacceptable acetate-containing buffer. A preferred vial volume is onesuitable for multi-dose use—allowing repeated withdrawal of sample. Theincreased stability attained with the formulations of the inventionallow multi-dose liquid formulation. Typically a multi-dose vial willprovide sufficient formulation to supply sufficient dosage for onepatient for one month, preferably one week. For example, the compositionvolume generally ranges from 0.3 to 10.0 ml and more preferably from 1.6to 2.0 ml, depending on dose concentration, frequency and ease of use.For example, a volume of 1.8 ml is convenient when either 0.3 ug/kg or0.1 ug/kg are used, allowing 7 or 24 doses, respectively. When a lightsensitive component, such as benzyl alcohol is present, the vial isprotected from intense light. Generally it is sufficient to store thevial in a darkened refrigerator or within an opaque box. However, thevial walls can comprise light transmission reducing materials. Forexample, translucent amber or brown vials or an opaque vial can be used.In preferred embodiments the vial contains multi-dose formulation. For avial configuration, a selected multi-dose liquid formulation can befilled in 3 cc Type I glass vial with 1.8 mL fill volume. Selection ofstopper will be based on compatibility of different types of stopperwith the selected formulation.

Compositions of the invention are typically stored at 2 to 8 degrees C.The formulations are stable to numerous freeze thaw cycles as shownherein.

In another embodiment the formulation is prepared with the above acetateconcentrations. A preferred means of preparing a formulation is todialyze a bulk NGF variant solution into the final formulation buffer.Final NGF variant concentrations are achieved by appropriate adjustmentof the formulation with formulation buffer absent NGF variant.

The compositions hereof including lyophilized forms, are prepared ingeneral by compounding the components using generally availablepharmaceutical compounding techniques, known per se. Likewise, standardlyophilization procedures and equipment well-known in the art areemployed. A particular method for preparing a pharmaceutical compositionof NGF variant hereof comprises employing purified (according to anystandard protein purification scheme) NGF variant, preferably rhNGFvariant, in any one of several known buffer exchange methods, such asgel filtration or dialysis.

The NGF variant-encoding gene constructs discussed herein can beinserted into target cells using any method known in the art, includingbut not limited to transfection, electroporation, calcium phosphate/DEAEdextran methods, and cell gun. The constructs and engineered targetcells can be used for the production of transgenic animals bearing theabove-mentioned constructs as transgenes, from which NGFvariant-expressing target cells may be selected using the methodsdiscussed. Alternatively, NGF variant can be delivered by gene therapyusing NGF variant-encoding nucleic acid. Selective expression ofrecombinant NGF variant in appropriate cells can be achieved using NGFvariant genes controlled by tissue specific or inducible promoters or byproducing localized infection with replication defective virusescarrying a recombinant NGF variant gene, such as certain adenoviruses asis known in the art.

In gene therapy applications, genes are introduced into cells in orderto achieve in vivo synthesis of a therapeutically effective geneticproduct, for example for replacement of a defective gene. “Gene therapy”includes both conventional gene therapy where a lasting effect isachieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology, 11:205-210 (1993)).Although, naked DNA is effective. In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87:3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science, 256:808-813 (1992).

The bioavailability of NGF is superior to that of NT-3. Accordingly, theinvention provides neurotrophic molecules with the activity of NT-3 andthe superior bioavailability of NGF. In addition, the invention providesneurotrophic molecules having unexpectedly superior pan-neurotrophicactivity compared to previous factors, thus having the additionaladvantage of obviating any problems associated with a mixture ofindividual neurotrophins.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.The disclosures of all citations in the specification are expresslyincorporated herein by reference.

EXAMPLES Example I Design and Synthesis of NGF Variants that Bind totrkC

Design of NGF Variants. The complete mutational analysis of human NT-3led to a detailed view of the binding epitope of NT-3 for its receptortrkC (Urfer et al., 1994). The binding site is dominated by a singleresidue, R103 (FIG. 3A). Analysis of the structural vicinity of R103revealed additional residues important for the NT-3/trkC interaction:K81 and Q84 on β-strand C, T23 on the loop connecting β-strands A and A′and, with smaller effects, the two conserved residues E55 and R57 onβ-strand B (FIGS. 2, 3A). In mouse NGF, T81 and H84, the residuesanalogous to NT-3 K81 and Q84 (FIGS. 2, 3B), have been shown to beinvolved in NGF binding to trkA (Ibáñez et al., 1993) and it istherefore possible that they contribute to the specificity. In humanNGF, V18, G23, T81, and H84 have been shown to be involved in trkAbinding. The other non-conserved NT-3 residue, T23, is located in anarea that is conserved within each member of the neurotrophins acrossspecies but is divergent between NT-3, BDNF and NGF. Near T23 andlocated on the same face of the molecule are E18 and L20 (FIG. 3A).Although they are not directly involved in binding to trkC, theirstructurally very different counterparts in NGF (V18 and V20) (FIG. 3B)and BDNF (18 and E20) may prevent binding to trkC. This suggested thatthe NGF residues V18, V20, G23, T81, and H84 and their respectivecounterparts in NT-3 are involved in determining specificity for trkC(Urfer et al., 1994). Therefore, variants of NGF that carried the NT-3amino acids at these positions were constructed and analyzed forrecruitment of trkC binding.

Synthesis of NGF Variants. NGF and NT-3 were previously cloned,sequenced and subcloned into a vector which allows for production ofdouble and single-stranded DNA in E. coli, as well as expression of theneurotrophins in a mammalian system under control of the cytomegaloviruspromoter (Rosenthal et al., 1990). Mutagenesis on this vector wasperformed according to the method of Kunkel (Kunkel, 1985). Aftertransformation into the E. coli strain XL1-Blue (Stratagene, San Diego,Calif.), colonies were screened for the presence of the desired mutationby sequencing double-stranded DNA using the Sequenase version 2.0 kit(U. S. Biochemical Corp., Cleveland, Ohio). The entire DNA sequencecoding for the mature NGF and NGF variants was verified for all positiveclones. Double-stranded DNA was isolated from XL-1 Blue with the QIAGENDNA purification kit (Qiagen Inc., Chatsworth Calif.).

Expression of wild-type and variant neurotrophins. Plasmid DNAcontaining either the NGF or variant coding sequences was introducedinto the human fetal kidney cell line 293 by calcium phosphateprecipitation (Gorman et al., 1990). The 75% confluent cells wereco-transfected with 10 μg of plasmid DNA and 1 μg of AdVA plasmid per 15mm cell culture dish and incubated for 15 h in serum-containing medium.Then the medium was removed and exchanged with serum-free medium (PSO4)supplemented with 10 mg/L recombinant bovine insulin, 1 mg/L transferrinand trace elements. Supernatant was collected after 48 and 96 h,concentrated approximately 20-fold with Centriprep-10 filtration units(Amicon, Beverly, Mass.) and sterile filtered.

Quantification of neurotrophin variants. The specific NGF ELISA(Enzyme-linked immunosorbent assay) was based on a Protein A-purifiedpolyclonal antiserum from guinea pig (Genentech, Inc.) and followedstandard ELISA procedures. A polyclonal serum was used in order toreduce the potential for differential cross-reactivity of variants tothe antibodies. The standard curve was determined using purifiedrecombinant NGF.

The amounts of NGF variants after concentration varied between 0.3 μg/mland 30 μg/ml. The ELISA assay did not detect any NGF in supernatantsfrom mock transfected cells. For each set of expressions of NGFvariants, a wild-type NGF expression was performed and quantified byELISA in parallel in order to obtain a comparative wild typeconcentration for receptor binding studies. All variants were expressed,quantified and assayed at least twice.

Example II Mutations in the Central β-strand Bundle of NGF Result inVariants that Bind to trkC

The variants NGF1 and NGF2 carried the mutations T81K/H84Q andG23T/V18E/V20L, respectively. The five point mutations were combined inthe variant NGF12. These three variants, as well as NGF and NT-3, wereexpressed and assayed for their ability to bind to the trkCextracellular domain.

Receptor immunoadhesin proteins were constructed using human trkA andtrkC extracellular domains fused to immunoglobulin constant domains(Shelton et al., 1995). Binding assays were performed as described(Shelton et al., 1995) using a 96-well plate format. The finalconcentration of labeled neurotrophin in each well was approximately 30pM for trkA and trkC binding assays. Purified recombinant human NT-3,BDNF and NGF (Genentech) were iodinated as described (Urfer et al.,1994). Usually, 20 μg of the neurotrophins were iodinated to specificactivities ranging from 2000-3000 Ci/mmol. The labeled material wasstored at 4° C. and used within 2 weeks of preparation. Variants wereassayed for binding affinity to the trkA and trkC receptor at leasttwice for each of the multiple expressions. This procedure allowedestimation of the error in affinity determination for each of thevariants. All data were analyzed by applying a four-parameter fitprocedure on the data set with the Kaleidagraph software package.Binding results in Table 3 are expressed as IC50mut/IC50 wt ratio. IC50is the concentration of variant resulting in 50% inhibition of bindingof native neurotrophin.

In competitive displacement binding assays, the NT-3 wild type displayedan affinity of 21.0±4.9 pM for trkC while NGF bound to this receptorwith an affinity reduced by 3587-fold compared to NT-3 (Table 3). Thevariants NGF1 and NGF2 bound to trkC with 1036-fold and 291-fold loweraffinity than NT-3, respectively (Table 3); this represents gains ofaffinity to trkC, when compared to NGF, of 3.5-fold and 12-fold,respectively. When the mutations in NGF1 and NGF2 were combined in thevariant NGF12 the affinity to trkC was substantially increased in asynergistic manner. This variant bound to trkC with only 14.7-foldreduced affinity compared to NT-3 which represents a 244-fold increaseof affinity when compared to NGF (Table 3).

TABLE 3 Relative affinities of NGF variants to trkC and trkA. ReceptortrkA trkC trkC Residue Number IC50_(mut) IC50_(mut) IC50_(mut) Variant18 20 23 29 79 81 84 86 IC50_(NGF) IC50_(NT-3) IC50_(NGF12) NGF V V G TY T H F 1.0 ± 0.1 3587 ± 771  (SEQ ID NO:1) NT-3 E L T I Q K Q Y 137 ±43  1.0 ± 0.2 (SEQ ID NO:2) NGF1 V V G T Y K Q F 0.7 ± 0.2 1036 ± 184 (SEQ ID NO:3) NGF2 E L T T Y T H F 2.1 ± 0.6 291 ± 75  (SEQ ID NO:4)NGF12 E L T T Y K Q F 1.5 ± 0.7 14.7 ± 4.8  1.0 (SEQ ID NO:5) NGFR1 E LG T Y K Q F 1.1 ± 0.2 3560 ± 875  242 (SEQ ID NO:17) NGFR2 V L T T Y K QF 0.9 ± 0.2 10.5 ± 2.5  0.7 (SEQ ID NO:18) NGFR3 E V T T Y K Q F 1.6 ±0.2 37.2 ± 10.1 2.5 (SEQ ID NO:19) NGFR4 E L T T Y T Q F 1.5 ± 0.3 14.8± 5.1  1.0 (SEQ ID NO:21) NGFR5 E L T T Y K H F 2.2 ± 0.4 113 ± 40  7.7(SEQ ID NO:20) NGF123 E L T I Y K Q F 1.2 ± 0.1 17.9 ± 7.3  1.2 (SEQ IDNO:6) NGF124 E L T T Q K Q Y 1.1 ± 0.2 7.8 ± 2.0 0.5 (SEQ ID NO:7)NGF125 E L T T Y K Q F 1.2 ± 0.3 14.0 ± 5.4  0.9 (SEQ ID NO:8) +F54Y/K57R NGF1234 E L T I Q K Q Y 1.3 ± 0.2 4.9 ± 1.5 0.3 (SEQ ID NO:9)NGF126 E L T T Y K Q Y 1.6 ± 0.1 3.3 ± 0.8 0.2 (SEQ ID NO:10) NGF127 E LT T Q K Q F 1.0 ± 0.1 33.0 ± 6.0  2.2 (SEQ ID NO:11) NGF130 V L T T Y TQ Y 1.0 ± 0.1 4.5 ± 0.7 0.3 (SEQ ID NO:12) NGF131 V L T I Y T Q Y 1.1 ±0.1 3.3 ± 0.9 0.2 (SEQ ID NO:13) Affinities to trkA and trkC are shownrelative to NGF and NT-3, respectively. The IC50 values were 33.9 ± 7.5pM (n = 12) for NGF binding to trkA and 21.0 ± 4.9 pM (n = 16) for NT-3binding to trkC. The results for variant affinities are expressed as theaverage of at least four independent binding experiments using proteinsfrom two different expressions ± SD. NT-3 residues are shown in italic.

Example III NGF Variants with Mutations in the Central b-strand BundleRetain trkA Binding

All NGF variants, NGF and NT-3 were assayed for trkA binding. NGFdisplayed an affinity of 33.9±7.5 pM while NT-3 bound with 137-foldreduced affinity compared to NGF (Table 3). This reduction in affinityis in agreement with earlier results (Urfer et al., 1994). The variantsNGF1, NGF2 and NGF12 bound to trkA with 0.7-fold, 2.1-fold and 1.5-foldreduced affinity, respectively (Table 3). This demonstrates that thechanges in NGF2 resulted in a slight loss of affinity to trkA whilechanges in NGF1 led to a small increase in affinity. When NGF1 and NGF2were combined in NGF12 the affinity of NGF12 to trkA was additive; incontrast the affinity of NGF12 to trkC was synergistic when compared toNGF1 and NGF2 (Table 3).

Example IV Importance of Individual Residues for trkC Specificity

In order to determine the importance for specificity of individualresidues that were changed in NGF12, each of these residues was changedback to the NGF sequence. Variants NGFR1, NGFR2, NGFR3, NGFR4 and NGFR5(Table 3) tested the contribution to specificity of G23, V18, V20, T81and H84, respectively. Variant NGFR1 lost most of its ability tointeract with trkC, NGFR3 and NGFR5 had their affinities significantlyreduced, while NGFR2 and NGFR4 had affinities to trkC similar to NGF12(Table 3). These data suggested that the most important specificitydeterminants for trkC binding are T23/G23, Q84/H84 and L20/V20(NT3/NGF).

Example V NGF12 Variants with Increased Affinity to trkC

Comparison of the model of human NT-3 (Urfer et al., 1994) and the X-raystructure of mouse NGF (McDonald et al., 1991) revealed several residuesclose to the main specificity determinants that differ between the twomolecules. In order to further increase the affinity of NGF12 to trkC,some of these residues were changed to the analogous NT-3 amino acids.Adding the changes F54Y/K57R to NGF12 did not improve trkC binding(NGF125, Table 3). In contrast, addition of Y79Q/F86Y did enhancebinding 2-fold compared to NGF12 (NGF124, Table 3). Individuallychanging these two residues in the NGF12 background showed that the F86Yexchange was a beneficial one while the Y79Q exchange was detrimental(NGF126 and NGF127, Table 3). Finally, the change T29I was evaluated. Inthe NGF12 background, T29I effected a slight decrease in binding(compare NGF12 versus NGF123, Table 3) but in the NGF124 and NGF126backgrounds it slightly improved binding (compare variants NGF124 versusNGF1234 and NGF130 versus NGF131, Table 3). This may be due tointeraction of the sidechains at positions 29, 86 and 103 since residue103 is situated between residues 29 and 86 (FIG. 3). Alternatively,sidechains at positions 29 and 86 may interact with the same amino acidin trkC and thereby influence one another via the trkC amino acid.

Based on these data, two additional variants were designed. VariantNGF31 contained the five changes V20L, G23T, V18E, T29I, H84Q and F86Yand bound to trkC with an affinity that is only 3.3-fold reducedcompared to NT-3. These changes did not affect trkA binding (Table 3).NGF126 and NGF131 bind equally well to trkC and trkA. Both variants havethe mutations V20L, G23T, H84Q and F86Y in common; NGF126 has theadditional mutations V18E and T81K while NGF131 has the additionalmutation T29I. This suggests that the changes V20L, G23T, H84Q and F86Yin NGF are the minimum required for recruitment of trkC binding. Indeed,NGF130, which possesses these four mutations, bound to trkC similar toNGF126 and NGF31 (Table 3).

Example VI NGF Variants Induce trkC Receptor Autophosphorylation

The ability of NGF variants to stimulate trk receptorautophosphorylation on PC12 cell lines was determined. Approximately1×10⁷ PC12 cells (Tsoulfas et al., 1993) were treated at 37° C. for 5min with 100 ng/ml neurotrophin. NP-40 plate lysis andimmunoprecipitation with an anti-trkA specific polyclonal antiserum(from Dr. Louis Reichardt, University of California, San Francisco) oranti-trkC specific polyclonal antiserum 656 (Urfer et al., 1994) wasperformed as previously described (Tsoulfas et al., 1993). Thephosphotyrosine content was analyzed by Western blot using monoclonalantibody 4G10 as previously described (Soppet et al., 1991; Tsoulfas etal., 1993). All tyrosine autophosphorylation assays were performed atleast twice for each neurotrophin assayed.

PC12 cells that were engineered to constitutively express rat trkCrespond to NT-3 by induction of strong autophosphorylation of trkC andformation of neurite extensions (Tsoulfas et al., 1993). Purified NGF(NGF/P) as well as supernatant of NGF-expressing 293 cells (NGF/U)resulted in a strong signal for trkA autophosphorylation (FIG. 4A) butdid not induce autophosphorylation of trkC (FIG. 5A). Purified NT-3(NT-3/P) and supernatant of NT-3-expressing 293 cells (NT-3/U) inducedautophosphorylation of trkC (FIG. 5A) but not trkA (FIG. 4A). Asexpected from the affinity of NGF12 for trkC, this variant resulted in astrong signal for trkC autophosphorylation (FIG. 5A) while maintainingits ability to elicit autophosphorylation of trkA (FIG. 4A). The ratherlow affinity for trkC of the variants NGF1 and NGF2 is reflected in theweak signal in trkC autophosphorylation (FIG. 5A). However, bothvariants still induced autophosphorylation of the trkA receptor withonly slightly reduced activity when compared to NGF (FIG. 4A).

Variants NGFR1 and NGFR5 were assayed for induction ofautophosphorylation of PC12/trkC cells and NGFR1 resulted in a very weaksignal while the NGFR5 response was between that of NGF12 and NGFR1(FIG. 5B). These results correlate with the determined affinities of thevariants for trkC. Variants of NGF12 (NGF123, NGF124, NGF125 andNGF1234) that further increased the affinity to trkC resulted in signalsfor trkC autophosphorylation that were similar to that elicited by NT-3(FIG. 5B). The three variants which bound best to trkC—NGF126, NGF130and NGF131—also elicited strong signals for autophosphorylation of trkC(FIG. 5C) as well as of trkA (FIG. 4B). These results demonstrate thatthe NGF variants that had increased affinity to trkC were also able tointeract with this receptor in the context of a model neuronal-like cellline. Furthermore, it is important to note that these cells also expresstrkA and that both receptors (trkA and trkC) compete for themultifunctional ligands. While it is possible that the NGF variantscould elicit formation of a trkA/trkC heterodimer, this might not leadto transphosphorylation of the two receptors (Canossa et al., 1996).

Example VII NGF Variants Induce trkC Cellular Signaling

The ability of NGF variants to stimulate differentiation of PC12 cellsexpressing trkC was determined. This indicates the ability of a variantto induce trkC cellular signaling. Approximately 10³ PC12 cellsexpressing trkC (Tsoulfas et al., 1993; Tsoulfas et al., 1996) wereplated onto 35 mm collagen-coated tissue culture dishes containing atotal of 2 ml of medium. PC12/trkC cells were assayed at neurotrophinconcentrations ranging from 250 pg/ml to 100 ng/ml. The proportion ofneurite-bearing cells was determined by counting the number of cellscontaining processes at least twice the length of the cell body after 3days. All neurite extension assays were performed at least twice.

Results from induction of neurite outgrowth in PC12/trkC cells areconsistent with the binding and autophosphorylation assays (Table 4).While PC12/trkC cells possess both trkA and trkC, it has been shownpreviously that NT-3 leads to a significant induction of neurites duringthe first three days after application of neurotrophin whereas NGF doesnot; however, at ten days NGF and NT-3 induce similar neurite outgrowth(Tsoulfas et al., 1996). Hence the PC12/trkC cells were evaluated forneurites after three days. Those variants which showed reduced bindingto trkC also showed reduced response. NGF12 exhibited a dose-responsewhich was shifted to higher values compared to native NT-3 and thosevariants with the poorest binding (NGF1, NGF2, NGFR1 and NGFR5 ) did notreach maximal response even at the highest dose tested (Table 4). NGF126was as potent in neurite induction as native NT-3 (Table 4) and thoughvariants NGF126, NGF130 and NGF131 were equivalent in trkC binding(Table 3), NGF126 was more efficacious in inducing neurites, reachingmaximal response at 1 ng/ml compared to 10 ng/ml for NGF130 and NGF131(Table 4). Notably, these NGF variants could induce neurites throughtrkC in an environment where trkA competes with trkC for their binding.At ten days, the NGF12, NGF1 and NGF2 variants acted similarly as NGF ininducing neurites (data not shown) as would be expected from the bindingof these variants to trkA (Table 3).

TABLE 4 Induction of Neurite Outgrowth in PC12 Cells Expressing Rat trkCConcentration of Neurotrophin in Medium Variant 250 pg/ml 1 ng/ml 10ng/ml 100 ng/ml NT-3  46 ± 6^(a) 74 ± 4 73 ± 5 71 ± 11 NGF 0 0 0 0 NGF10 0 0 9 ± 1 NGF2 14 ± 3 35 ± 8 42 ± 9 48 ± 12 NGF12 16 ± 8 25 ± 6 54 ± 959 ± 7  NGFR1  4 ± 1  3 ± 2  7 ± 1 13 ± 5  NGFR5  5 ± 3 13 ± 5 23 ± 5 46± 16 NGF126 35 ± 6 70 ± 9 72 ± 3 69 ± 10 NGF130 26 ± 4 46 ± 3 69 ± 5 71± 4  NGF131 20 ± 6 49 ± 7 68 ± 2 67 ± 7  ^(a)Values are the percent ofcounted cells that carried neurites which were at least twice the lengthof the cell body.

Discussion

The neurotrophins transduce their signal into the cell by interactionwith the trk receptor tyrosine kinases (Kaplan & Stephens, 1994). Theneurotrophins and the trks both form highly homologous protein families.Within each family the different members probably have similarstructures, but individual members of the two families interact witheach other in a very specific manner. This inherent specificity ofneurotrophins is necessary for their biological function and thereforeinformation on the mechanisms of specificity determination contributesto an understanding of function and evolution of the neurotrophinfamily.

Molecular modeling and alanine scanning mutagenesis of human NT-3 (Urferet al., 1994) and domain deletions/swaps of the human trks (Urfer etal., 1995) determined the binding epitopes of this ligand/receptorsystem. The former study revealed that the binding site of NT-3 for itsreceptor trkC is dominated by residue R103, with additional determinantsin its vicinity. The binding site extends around the central β-strandbarrel and, in contrast to the NGF binding site for trkA, does notinclude residues from loops and the first six residues of the N-terminus(FIG. 3). Non-conserved residues that are part of the binding siteinclude T23, K81 and Q84. Residue T23, together with L18 and E20, arelocated in an area which is conserved across all species within each ofthe members of the neurotrophin family, but is divergent between NT-3,NGF and BDNF. Therefore, these five residues seemed to be reasonablecandidates for specificity determinants in NT-3 for trkC binding. Inorder to test their importance for binding to trkC, residues in NGF(V18, V20, G23, T81 and H84) were changed to their corresponding NT-3amino acids (E18, L20, T23, K81 and Q84) and the resulting protein,NGF12, was analyzed for recruitment of trkC binding and trkC mediatedbiological activities. NGF12 was able to bind to trkC, induceautophosphorylation of trkC expressed on PC12 cells and did not loseaffinity to trkA (Table 3, FIGS. 4, 5).

As indicated herein the change from Glycine23 to Threonine dominates therecruitment effect in NGF12. Additional variants of NGF12 in which eachof the mutated residues was individually changed back to the originalNGF amino acid (i.e. E18V, L20V, T23G, K81T, Q84H) demonstrated that inNT-3 the most important determinant for trkC specificity is T23 followedby Q84 and L20. The change from glycine to threonine in NGF mayintroduce a sidechain that is critical in binding to trkC;alternatively, the G23T change might effect a change of the backboneconformation and thereby influence the conformation of sidechains in itsvicinity. At position 84 the change from histidine to glutamine removesa potentially charged residue which might be repulsive to trkC or theglutamine sidechain may be required for specific hydrogen bonding.Finally, the preference for leucine over valine at position 20 may bedue to rather stringent spatial requirements of a hydrophobicinteraction at the binding site.

In addition to T23, Q84 and L20, the amino acid at position 86 isimportant for neurotrophin specificity to trkC. Adding the NT-3 residueto NGF12 effected a 4.5-fold improvement in binding to trkC whilemaintaining binding to trkA (variant NGF126, Table 3). Residue 86 isproximal to the most important determinant for binding to trkC, R103(FIG. 3), further confirming the dominance of this structural region fortrkC recognition. In NGF residue 86 is a phenylalanine and in NT-3 it isa tyrosine suggesting that the sidechain hydroxyl group may be involvedin a specific hydrogen bond required for trkC recognition.

Finally, residues at positions 18 and 29 may fine tune the specificityof NT-3 for trkC. The effect at position 29 seems to be dependent on thecharacter of the amino acid at position 86. When the change T29I wasintroduced into NGF12 (which has F86), trkC binding was slightly reducedwhereas when T29I was introduced into two other variants (which haveY86) the binding was slightly improved (compare NGF124 versus NGF1234,NGF130 versus NGF131, Table 3). Similar to residue 86, residue 29 isproximal to the important R103 (FIG. 3). At position 18, the mutationV18E contributed only slightly to trkC binding (cf. NGF12 and NGFR2,Table 3). However, comparison of induction of neurite outgrowth byNGF126 and NGF130 shows that inclusion of the V18E mutation improvedinduction of neurite outgrowth; NGF126, which includes V18E, elicitedneurite outgrowth equivalent to native NT-3 (i.e. reaching maximalresponse at 1 ng/ml) whereas NGF130 required 10-fold more neurotrophinto elicit the maximal response (10 ng/ml) (Table 4).

In summary, the major specificity determinants on NT-3 for trkC includeL20, T23, Q84 and Y86. Residues E18 and I29 may play a minor role inspecificity. The six residues form two clusters: I29, Q84 and Y86 areproximal to R103, the most important residue involved in trkC binding,and E18 and T23 are located together but distant from the R103 cluster(FIG. 3). This suggests that trkC uses at least two unique, spatiallydistant sites to discriminate between the various neurotrophins.

Earlier mutational analyses of rodent and human neurotrophins proposedthat the first few residues of the N-terminus of NGF are important forspecific binding to trkA (Ibáñez et al., 1993; Urfer et al.; 1994, Shihet al., 1994). NT-3 variants that carried the N-terminal residues fromNGF were constructed and the resulting molecules MNTS-1 and PNT-1 indeedacquired affinity to trkA similar to NGF (Urfer et al., 1994; Ibáñez etal., 1993). This implies that the specificity of neurotrophin binding totrkA is dominated by the N-terminal residues and that the remainder ofthe NT-3 molecule is fully compatible with binding to trkA. Furthermore,the observation that MNTS-1 did not lose its affinity to trkC impliesthat the N-terminal residues of NT-3 are not involved in trkCrecognition, a conclusion that was corroborated by site-directedmutagenesis of the NT-3 N-terminus (Urfer et al., 1994). The presentwork establishes the importance of residues located in the centralβ-strand bundle for function and specificity of NT-3. Non-conservedamino acids of NT-3 that were previously shown to be involved in trkCbinding by site-directed mutagenesis (Urfer et al., 1994)—T23 andQ84—can be substituted in NGF and recruit trkC binding. In addition,non-conserved residues in NT-3—E18, L20, I29, and Y86—which apparentlydo not contribute to the binding of NT-3 to trkC (Urfer et al., 1994)can also contribute to the specificity of the NT-3/trkC interaction,though exhibiting a reduced effect compared to T23 and Q84. Replacingthese six residues in NGF with their NT-3 counterparts maintains fullaffinity to trkA suggesting that trkA does not utilize these specificamino acid positions to discriminate between NGF and NT-3. Hence, inNT-3 residues in the central β-strand bundle impart specificity whereasin NGF the N-terminal residues impart specificity.

Based on sequence conservation in NGF from different species, an earlierstudy proposed that Y79, T81, and H84 could be mediating the interactionof NGF with trkA (Ibáñez et al., 1993). This same study also found thatreplacing these three residues in NGF with those from BDNF (Q79, R81,Q84) resulted in a chimeric neurotrophin that could activate trkB. Incontrast, replacing Y79Q and T81R did not activate trkB (Ibáñez et al.,1993), suggesting that only residue 84 is important for trkB activation.In the present study, it was determined that residue 81 did not affectbinding or specificity for trkA or trkC (cf. NGF12 and NGFR4, Table 3)and residue 84 was important for trkC specificity but not for trkAspecificity (cf. NGF12 and NGFR5, Table 3). The present study alsodetermined that NGF residue 79 was not involved in trkA specificitywhereas for trkC the native human NGF residue (Y79) was slightlypreferred compared to the human NT-3 Q79 (NGF127, Table 3). Takentogether, the data from these two studies suggest that position 84 playsa role in specificity for trkB and trkC, but not trkA.

The results presented here show that grafting a specific set of centralβ-strand bundle residues from human NT-3 onto human NGF could recruitbinding to the non-cognate receptor (trkC) while maintaining theaffinity for the cognate receptor (trkA). Therefore, the determinantsfor specificity of the neurotrophins to the different trk receptors arenot only located in homologous positions in loop regions as proposedpreviously (Ilag et al., 1994; Ryden & Ibáñez, 1996), but also involveresidues in the central β-strand bundle. Our data suggest a mechanism ofspecificity determination similar to that proposed for the homologousfamilies of gonadotropins and gonadotropin receptors (Moyle et al.,1994) where the luteinizing hormone receptor recognizes regions on theluteinizing hormone that are distinct from the ones that the homologousfollitropin receptor recognizes on follitropin.

Due to the high sequence homologies within the neurotrophins and thetrks it is very likely that both families co-evolved from commonancestors. An ancestral neurotrophin may have used the central β-strandbundle region around R103 for binding. Neurotrophins with unique trkspecificities then could have diverged from the ancestral neurotrophinby acquiring repulsive and/or attractive forces on the neurotrophinsurfaces encompassing the N-terminus, the variable loops, and thecentral β-strand barrel. In this respect it is important to note thatthe specificity profile of a present day neurotrophin could be changedby a single point mutation (Urfer et al., 1994).

Administration of neurotrophins in models of nerve lesions have beenshown to be beneficial for regeneration and survival of neurons(Sendtner et al., 1992; Yan et al., 1992). Neuronal populations involvedin neurodegenerative disorders may express more than one trk receptorand therefore administration of molecules with multiple specificities,such as MNTS-1 (Urfer et al., 1994), PNT-1 (Ibáñez et al., 1993), andNGF126, could be advantageous compared to administration of a singlemonospecific neurotrophin or a cocktail of monospecific neurotrophins.For example, the various members of the neurotrophin family may havedifferent pharmacokinetics and therefore the behavior of neurotrophincocktails could be difficult to predict or control.

This application is related to U.S. Ser. No. 08/441,353, filed May 15,1995, now abandoned, which is a continuation of U.S. Ser. No. 08/253,937filed Jun. 3, 1994, now abandoned, both of which are incorporated hereinby reference.

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21 120 amino acids Amino Acid Linear 1 Ser Ser Ser His Pro Ile Phe HisArg Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Val Ser Val Trp Val GlyAsp Lys Thr Thr Ala Thr Asp 20 25 30 Ile Lys Gly Lys Glu Val Met Val LeuGly Glu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe GluThr Lys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly IleAsp Ser Lys His 65 70 75 Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe ValLys Ala Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile ArgIle Asp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val ArgArg Ala 110 115 120 119 amino acids Amino Acid Linear 2 Tyr Ala Glu HisLys Ser His Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser Glu Ser LeuTrp Val Thr Asp Lys Ser Ser Ala Ile Asp Ile 20 25 30 Arg Gly His Gln ValThr Val Leu Gly Glu Ile Lys Thr Gly Asn 35 40 45 Ser Pro Val Lys Gln TyrPhe Tyr Glu Thr Arg Cys Lys Glu Ala 50 55 60 Arg Pro Val Lys Asn Gly CysArg Gly Ile Asp Asp Lys His Trp 65 70 75 Asn Ser Gln Cys Lys Thr Ser GlnThr Tyr Val Arg Ala Leu Thr 80 85 90 Ser Glu Asn Asn Lys Leu Val Gly TrpArg Trp Ile Arg Ile Asp 95 100 105 Thr Ser Cys Val Cys Ala Leu Ser ArgLys Ile Gly Arg Thr 110 115 119 120 amino acids Amino Acid Linear 3 SerSer Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 AspSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp 20 25 30 Ile LysGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro ValAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Tyr CysLys Thr Thr Gln Thr Phe Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys GlnAla Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys ValLeu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120 120 amino acids AminoAcid Linear 4 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser ValCys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Thr Asp Lys Thr Thr Ala ThrAsp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 5055 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 7075 Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu 80 85 90Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120120 amino acids Amino Acid Linear 5 Ser Ser Ser His Pro Ile Phe His ArgGly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Thr AspLys Thr Thr Ala Thr Asp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu GlyGlu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu ThrLys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile AspSer Lys His 65 70 75 Trp Asn Ser Tyr Cys Lys Thr Thr Gln Thr Phe Val LysAla Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg IleAsp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg ArgAla 110 115 120 120 amino acids Amino Acid Linear 6 Ser Ser Ser His ProIle Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Glu Ser LeuTrp Val Thr Asp Lys Thr Thr Ala Ile Asp 20 25 30 Ile Lys Gly Lys Glu ValMet Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln TyrPhe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly CysArg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Tyr Cys Lys Thr Thr GlnThr Phe Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp ArgPhe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg LysAla Val Arg Arg Ala 110 115 120 120 amino acids Amino Acid Linear 7 SerSer Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 AspSer Glu Ser Leu Trp Val Thr Asp Lys Thr Thr Ala Thr Asp 20 25 30 Ile LysGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro ValAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Gln CysLys Thr Thr Gln Thr Tyr Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys GlnAla Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys ValLeu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120 120 amino acids AminoAcid Linear 8 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser ValCys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Thr Asp Lys Thr Thr Ala ThrAsp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Arg Asp 5055 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 7075 Trp Asn Ser Tyr Cys Lys Thr Thr Gln Thr Phe Val Lys Ala Leu 80 85 90Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120120 amino acids Amino Acid Linear 9 Ser Ser Ser His Pro Ile Phe His ArgGly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Thr AspLys Thr Thr Ala Ile Asp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu GlyGlu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu ThrLys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile AspSer Lys His 65 70 75 Trp Asn Ser Gln Cys Lys Thr Thr Gln Thr Tyr Val LysAla Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg IleAsp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg ArgAla 110 115 120 120 amino acids Amino Acid Linear 10 Ser Ser Ser His ProIle Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Glu Ser LeuTrp Val Thr Asp Lys Thr Thr Ala Thr Asp 20 25 30 Ile Lys Gly Lys Glu ValMet Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln TyrPhe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly CysArg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Tyr Cys Lys Thr Thr GlnThr Tyr Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp ArgPhe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg LysAla Val Arg Arg Ala 110 115 120 120 amino acids Amino Acid Linear 11 SerSer Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 AspSer Glu Ser Leu Trp Val Thr Asp Lys Thr Thr Ala Thr Asp 20 25 30 Ile LysGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro ValAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Gln CysLys Thr Thr Gln Thr Phe Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys GlnAla Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys ValLeu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120 120 amino acids AminoAcid Linear 12 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser ValCys 1 5 10 15 Asp Ser Val Ser Leu Trp Val Thr Asp Lys Thr Thr Ala ThrAsp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 5055 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 7075 Trp Asn Ser Tyr Cys Thr Thr Thr Gln Thr Tyr Val Lys Ala Leu 80 85 90Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120120 amino acids Amino Acid Linear 13 Ser Ser Ser His Pro Ile Phe His ArgGly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Val Ser Leu Trp Val Thr AspLys Thr Thr Ala Ile Asp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu GlyGlu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu ThrLys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile AspSer Lys His 65 70 75 Trp Asn Ser Tyr Cys Thr Thr Thr Gln Thr Tyr Val LysAla Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg IleAsp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg ArgAla 110 115 120 6 amino acids Amino Acid Linear 14 Ser Lys Lys Arg IleGly 1 5 6 7 amino acids Amino Acid Linear 15 Ser Ser Ser His Pro Ile Phe1 5 7 6 amino acids Amino Acid Linear 16 Tyr Ala Glu His Lys Ser 1 5 6120 amino acids Amino Acid Linear 17 Ser Ser Ser His Pro Ile Phe His ArgGly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Gly AspLys Thr Thr Ala Thr Asp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu GlyGlu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu ThrLys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile AspSer Lys His 65 70 75 Trp Asn Ser Tyr Cys Lys Thr Thr Gln Thr Phe Val LysAla Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg IleAsp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg ArgAla 110 115 120 120 amino acids Amino Acid Linear 18 Ser Ser Ser His ProIle Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Val Ser LeuTrp Val Thr Asp Lys Thr Thr Ala Thr Asp 20 25 30 Ile Lys Gly Lys Glu ValMet Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln TyrPhe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly CysArg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Tyr Cys Lys Thr Thr GlnThr Phe Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp ArgPhe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg LysAla Val Arg Arg Ala 110 115 120 120 amino acids Amino Acid Linear 19 SerSer Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 AspSer Glu Ser Val Trp Val Thr Asp Lys Thr Thr Ala Thr Asp 20 25 30 Ile LysGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn 35 40 45 Asn Ser ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 50 55 60 Pro Asn Pro ValAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 70 75 Trp Asn Ser Tyr CysLys Thr Thr Gln Thr Phe Val Lys Ala Leu 80 85 90 Thr Met Asp Gly Lys GlnAla Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105 Thr Ala Cys Val Cys ValLeu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120 120 amino acids AminoAcid Linear 20 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser ValCys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Thr Asp Lys Thr Thr Ala ThrAsp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp 5055 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His 65 7075 Trp Asn Ser Tyr Cys Lys Thr Thr His Thr Phe Val Lys Ala Leu 80 85 90Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp 95 100 105Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg Ala 110 115 120120 amino acids Amino Acid Linear 21 Ser Ser Ser His Pro Ile Phe His ArgGly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Glu Ser Leu Trp Val Thr AspLys Thr Thr Ala Thr Asp 20 25 30 Ile Lys Gly Lys Glu Val Met Val Leu GlyGlu Val Asn Ile Asn 35 40 45 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu ThrLys Cys Arg Asp 50 55 60 Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile AspSer Lys His 65 70 75 Trp Asn Ser Tyr Cys Thr Thr Thr Gln Thr Phe Val LysAla Leu 80 85 90 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg IleAsp 95 100 105 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg ArgAla 110 115 120

What is claimed is:
 1. A nucleic acid comprising a nucleotide sequenceencoding an NGF variant having substitutions at amino acid positionsG23, H84, and V18 or V20 of SEQ ID NO: 1, so that the variant bindstrkC, said variant otherwise retaining the sequence of SEQ ID NO:
 1. 2.An expression vector comprising the nucleic acid of claim
 1. 3. Anisolated host cell comprising the nucleic acid of claim
 1. 4. A methodof producing an NGF variant, comprising culturing the host cell of claim3 under conditions that allow expression of the NGF variant.
 5. Themethod of claim 4, further comprising the steps of isolating the NGFvariant.
 6. The nucleic acid of claim 1, wherein V20 is substituted. 7.The nucleic acid of claim 1, wherein the NGF variant further comprises asubstitution of F86.
 8. The nucleic acid of claim 1, wherein the NGFvariant further comprises a substitution of T81.
 9. The nucleic acid ofclaim 1, wherein the NGF variant further comprises a substitution ofT29.
 10. The nucleic acid of claim 1, wherein the NGF variant isselected from the group consisting of NGF130 (SEQ ID NO:12), NGF131 (SEQID NO:13), NGFR2 (SEQ ID NO:18), and NGFR3 (SEQ ID NO:19).
 11. Thenucleic acid of claim 1, wherein the NGF variant further comprises anamino acid substitution at D16 which imparts trkB binding to the NGFvariant.
 12. The nucleic acid of claim 1, wherein the NGF variantfurther comprises a modification of a ten-amino-acid-N-terminal sequenceof NGF, that further reduces or eliminates trkA binding, wherein theN-terminal amino acid sequence SerSerSerHisProllePhe is absent.
 13. Thenucleic acid of claim 1, wherein at least one of the amino acids in theten-amino-acid-N-terminal sequence of the NGF variant are deleted orsubstituted to reduce or eliminate trkA binding.
 14. The nucleic acid ofclaim 12, wherein the NGF variant further comprises an amino acidsubstitution imparting trkB binding at D16.
 15. The nucleic acid ofclaim 1, wherein the NGF variant further comprises a deletion of aminoacid R119 or A120 or both.
 16. The nucleic acid of claim 15, wherein theNGF variant comprises a deletion of amino acid R118.
 17. A nucleic acidcomprising a nucleotide sequence encoding an NGF variant havingsubstitutions at amino acid positions V18, V20, G23, H84 and either orboth F86 or T81 of SEQ ID NO: 1, so that the variant binds trkC, saidvariant otherwise retaining the sequence of SEQ ID NO:
 1. 18. Anexpression vector comprising the nucleic acid of claim
 17. 19. Anisolated host cell comprising the nucleic acid of claim
 17. 20. A methodof producing an NGF variant, comprising culturing the host cell of claim19 under conditions that allow expression of the NGF variant.
 21. Themethod of claim 20, further comprising the steps of isolating the NGFvariant.
 22. The nucleic acid of claim 17, wherein both T81 and F86 aresubstituted in the NGF variant.
 23. The nucleic acid of claim 17,wherein the NGF variant further comprises a substitution of T29.
 24. Thenucleic acid of claim 17, wherein the NGF variant is selected from thegroup consisting of NGF126 (SEQ ID NO: 10), NGF1234 (SEQ ID NO: 9),NGF124 (SEQ ID NO: 7), NGF125 (SEQ ID NO: 8), NGF12 (SEQ ID NO: 5),NGF123 (SEQ ID NO: 6), and NGF127 (SEQ ID NO: 11).